INTRODUCTION : Rickettsial infections are known causes of illness and death worldwide. Evidence is
accumulating that these diseases especially scrub typhus and spotted fever are reemerging in India. As these diseases present as acute undifferentiated febrile illness, a high index of suspicion is essential in diagnosing this condition. This is further compounded by non-availability of specific tests to diagnose rickettsial infections in most centers. The less sensitive but inexpensive, Weil–Felix test, still remains the main stay of laboratory diagnosis/confirmation of these infections in India. Many clinicians still rely on dramatic response to doxycycline therapy for confirming rickettsial disease in doubtful cases. Presently at our centre, we are routinely offering the spotted fever and scrub typhus IgM ELISA in addition to the Weil –Felix test for diagnosis. We suspect many cases of rickettsial infection such as spotted fever and scrub typhus are missed as IgM antibodies are detectable by these assays after the first week of infection. AIM AND OBJECTIVES : Aim of the study: To determine the utility of serological and molecular techniques in the diagnosis of spotted fever group of rickettsial infections. Objectives of the study: A. To standardize a polymerase chain reaction (PCR) assay for the detection of spotted fever group rickettsia. B. To evaluate the utility of PCR and IgM ELISA in confirming the clinical diagnosis of spotted fever. C. Comparison of whole blood and skin biopsy of the rash (specimen) for detection of spotted fever group (SFG) rickettsia by PCR. D. Confirmation of PCR amplification by sequencing of three randomly selected PCR products of the two gene targets used. MATERIALS AND METHODS : Children above the age of 6 months and adults with fever (≥5 and ≤15 days) and rash were enrolled into the study, if found eligible as per the inclusion and exclusion criteria enumerated below. These subjects were included after evaluation by a dermatologist and after ruling out drug reactions and viral exanthems, by history and examination (A clinical proforma was also filled up as given in Appendix II at the time of sample collection. Informed consent was obtained from all individuals recruited. Inclusion criteria: 1. Age ≥ 6 months, 2. Fever ≥5 and ≤15 days, 3. Rash: non confluent maculopapular rash with or without purpura/purpura fulminans. Exclusion criteria: 1. Age ≤ 6 months, 2. Individuals with duration of fever less than 5 days or more than 15 days, 3. No rash. SUMMARY AND CONCLUSION ; 1. A nested PCR to detect gltA gene and 17-kDa gene of spotted fever group rickettsia was standardized. 2. Using the case definition, 42 cases were diagnosed as spotted fever. ELISA detected 37 cases where as 38 cases were positive by gltA PCR, of these two were detected only in blood. The 17-kDa antigen gene was detected in 23 of the 38 gltA PCR positive cases. 3. The gltA PCR and ELISA showed a sensitivity of 90.48% and 88.1% respectively. A specificity of 94.28%, 88.24% was observed for these two
assays. 4. Two false positive results by ELISA and one by skin biopsy gltA PCR were observed. 5. Sequencing of three amplified products for each of the targets confirmed PCR amplification of spotted fever DNA. 6. In the early phase of illness (5th day to 8th day) PCR has performed better, whereas ELISA gives 100% sensitivity from the 8th day onwards. 7. Whole blood PCR detection rates were low when compared with skin biopsy results by PCR. The better performance of skin biopsy is most likely due to the presence of larger amount of organisms in endothelial cells of the small blood vessels in comparison to those found in the peripheral circulation
